The present invention relates to a vacuum chamber and a vacuum system using the vacuum chamber for the directed transport of a substance, especially a fluid, and to its use in an apparatus for automatic plasmid preparation.
The past few years have seen an increase in the scale of efforts to obtain the complete genetic information of entire organisms. Beginning with the sequencing of a phage genome (bacteriophage T7: 38000 base pairs, bacteriophage xcex: 48514 base pairs) and continuing by way of the genome of Escherichia coil (4.2xc3x97106 base pairs) to the yeast Saccharomyces cerevisiae (2.3xc3x97107 base pairs) as the first representative of the eukaryotes, the number of base pairs to be sequenced has increased almost 600-fold. In the meantime, the human genotype with more than 3xc3x97109 base pairs has become the goal of these efforts in the xe2x80x9cHuman Genome Projectxe2x80x9d. The enormous quantities of DNA to be sequenced are barely manageable by the means and personnel available to laboratories hitherto. There is therefore a demand for new technologies that, for an acceptable financial outlay, are capable of bringing about a considerable increase in the throughput of samples in this research programme. Two mutually influencing strategies have come to light in the course of current development: on the one hand the miniaturisation of laboratory sequences and on the other hand the unsupervised automation of well-established laboratory procedures.
The miniaturisation of laboratory sequences has given rise to miniaturised electrophoresis analysers in which the separation of biomolecules on the basis of their charge and size is utilised. Such miniaturised electrophoresis analysers are obtained by means of microstructures in electrophoresis chips. Also available are miniaturised PCR machines, wherein during the polymerase chain reaction (polymerase chain reaction=PCR) DNA fragments up to 6 kilobases in size are amplified. Also known are miniaturised sample arrays and miniaturised detection systems. Miniaturised elements such as those described above can be combined to form larger units, so that a complete miniaturised laboratory unit is obtained.
On the other hand, for the automation of a laboratory it is not absolutely necessary to miniaturise routine procedures. It is likewise possible to design a robot system that completely or partially replaces the manual tasks of a human being in order to achieve an increase in sample throughput. The following manual tasks are typical of a laboratory preparation (with particular emphasis on plasmid preparation as a preliminary to PCR sequencing) and need to be carried out by suitable robots:
pipetting
transport of used material and of chemicals
suction of fluids through filters, membranes, permeable solids or the like
PCR reaction.
Current pipetting robots locate standard laboratory material on a work surface at defined positions and thus enable tested laboratory protocols to be set up. For example, using auxiliary robots it is possible for microtitre plates, pipette tips or reservoirs for buffer solutions etc. to be installed on such machines and, after use, removed from the workstation again. Thus, all the necessary pipetting steps preliminary to a PCR or a plasmid preparation can be executed in order that the product of that pipetting operation can then be introduced into a suitable machine for further preparation using a gripping robot.
The polymerase chain reaction (PCR) amplifies a DNA segment when it is enclosed between two defined primer sites. If equal amounts of primers are used, double-stranded DNA copies are produced by the PCR, whereas if one primer is used in excess then, in accordance with that excess, single-stranded copies of the amplified DNA are obtained. Both single-stranded and double-stranded DNA can be used for sequencing. In sequencing-intensive projects the DNA fragments to be analysed are cloned into plasmids which are then in the first instance present in a defined matrix of bacterial colonies (Escherichia Coli Blue) growing on agar. The subsequent taking up of the colonies from the matrix into culture tubes can also be automated. Over an incubation period (37xc2x0 C.) lasting about 12 hours the living bacterial clones then yield sufficient material to obtain in a preparation the plasmid copies necessary for sequencing. Obtaining such purified plasmids for sequencing is achieved, for example, by the QIAWELL 96 ultraplasmid purification procedure. Such plasmid preparation procedures include filtering operations in which a fluid has to be transported in a directed manner from one filter into at least one second filter and either also passes through that filter or is simply collected in a controlled manner.
The problem underlying the invention is therefore to provide an apparatus in which the directed movement of substances can be carried out automatically.
The present invention relates to a vacuum chamber for the directed transport of a substance, especially a fluid, there being installed in the vacuum chamber a permeable means and a collecting means, so that there are defined at least two vacuum regions that can be established independently of one another, namely a first vacuum region between the permeable means and the collecting means and a second vacuum region between the collecting means and the base of the vacuum chamber, and a vacuum can be generated in the two vacuum regions independently of one another so that the substance, especially the fluid, can be sucked from the first permeable means into the collecting means. Fluids are here to be understood as being gases, liquids, vapours and fumes.
The second means is preferably also permeable, so that by the application of a vacuum to the second region the fluid or liquid is sucked through the second means into the second region. The vacuum chamber according to the invention will generally have exactly two vacuum regions, but more than two vacuum regions are possible, for example when several filtrations are to be carried out one immediately after another.
Furthermore, the permeable means are formed by filter supports having a large number of filter elements, so that fluid can be transported in a defined manner from a particular filter element of the first filter support into a corresponding filter element of the second filter support in turn through the latter into the second lower region of the vacuum chamber. The collecting means are likewise collector supports having a defined number of collecting elements. In the case of a collector support, therefore, the substance, especially the fluid, is transported through the first filter support into the collector support.
Moreover, the vacuum chamber consists of a cover and a lower part, the lower part of the vacuum chamber having a shoulder for receiving the lower filter support. In addition, recesses for the gripper of the robot are provided in the side walls of the chamber in order that the filter plates or filter supports can be inserted and removed automatically. For the exact receiving and guidance of the filter supports or the collector support the lower part of the vacuum chamber has guide tabs having correcting bevels. The guide tabs preferably have two different bevel angles, the first bevel angle being about 30xc2x0 and the second bevel angle being about from 0xc2x0 to 2xc2x0. Furthermore, the guide edges of the wall with which the filter supports come into contact on insertion can be bevelled. Preferably the cover has a bevelled guide edge so that when the cover is put in place it is centred using the guide edge of the cover. The edge bevel angle is preferably 30xc2x0. The cover also has in the wall region recesses for the robot gripper and a supporting surface for the upper filter support.
The sealing material for the upper filter holder preferably has a hardness of about 20 Shore, the seal at the join between the cover and the lower part being formed by a combination of an O-ring and a resilient sealing strip, the O-ring providing a seal of about 60 Shore and the sealing strip of about 30 Shore. Sealing is also effected at the lower filter support using a rubber gasket having a hardness of 60 Shore.
The upper part has corresponding receiving means for receiving the guide tabs so that the cover is centred on the lower part by means of the guide tabs.
In a preferred embodiment, the filter supports have N pipe-shaped individual filters (N being especially 96) that are connected to form a filter support. The same applies to the collector support. In addition, there are mounted on the corresponding four corner pies of the two filter supports, or of the filter support and the collector support, spacer sleeves which, in addition to their function of defining the first vacuum region, also effect the vertical correction of misplacements of the lower filter support by engaging in centring shafts in the vacuum lower part. The spacer sleeves preferably have a partially cylindrical shape in order to allow the vacuum to act on the corner pies. In addition, the spacer sleeves can be bevelled so that an additional centring of the filter supports is achieved during insertion. A filter support may be in one piece consisting of a large number of filter elements or it may be composed of a large number of individual filter elements.
The length of the spacer sleeves is preferably so selected that the outlet tips of the upper filter support are located inside the pipes of the lower filter support or collector support, so that a controlled transport of the fluid through pipes or elements that correspond to one another is achieved. The outlet tips of the elements of the upper filter support are preferably located 1.5 mm inside the corresponding pipes of the filter elements or collecting elements of the corresponding lower support. As a result, contamination of non-corresponding elements is avoided.
Preferably the vacuum chamber and the spacer sleeves are manufactured from plexiglass of a suitable thickness, which allows visual monitoring. For industrial production, the vacuum chamber may consist of a cast plastics material, which allows economical manufacture. Injection-moulding processes and milling processes may also be used.
In the lower part of the vacuum chamber there are arranged a suction shaft for the first vacuum region and a suction shaft for the second vacuum region. Fluid passing through during a filtration procedure is removed directly from the vacuum chamber through the suction shaft of the second vacuum region.
The present invention relates also to a vacuum system having at least one vacuum pump and an electronically controlled valve for the lower chamber region, an electronically controlled valve for the middle chamber region, a valve for breaking the creeping vacuum in the lower chamber region and a vacuum trap arranged between the valves and the connection to the lower region of the vacuum chamber for receiving the waste volume. Each proportional valve may have its own controlling electronics system which can be actuated by the control software via a decoding apparatus of a PC.
The invention relates also to an apparatus for automatic plasmid preparation having a vacuum system for automating the directed transport of a substance, a pipetting robot and a gripping robot, wherein the gripping robot inserts the filter supports, after pipetting has been carried out by the pipetting robot, into the vacuum chamber and closes the cover and, after filtering, opens the chamber and removes the filter supports and conveys them to a further processing step. Such an apparatus is preferably controlled by a computer. It is also possible to work with only one robot which assumes the gripping and the pipetting functions.
The apparatus also has a dryer for filter supports, because in some preparation procedures the last preparation step is washing with alcohol, so that residual alcohol adhering to the last support has to be removed.
Advantageously the vacuum chamber is part of a larger robot system which can be used for supporting all current preparation methods of molecular biology processes. A modular design has therefore been created which enables the apparatus components of the apparatus for automatic preparation to be arranged to suit the particular problem being posed. Furthermore, the robot system used has no feedback, which means that no visual or other sensory monitoring of the current actual state is possible. All the movable components of the system must therefore be located in positions that are defined as exactly as possible. When components are moved by a robot arm, it is important that those moved components, when taken up by the robot again, are located at exactly defined positions. The vacuum chamber is, in addition, removable from the system as a module so that other modular systems for other methods can be inserted in its place. Therefore the position of all auxiliary systems for the automated preparation is oriented on the pipetting robot. In order, therefore, to be able to monitor the current position of the gripping robot visually during the xe2x80x9clearning phasexe2x80x9d of the system, the vacuum chamber is advantageously manufactured from plexiglass or some other transparent plastics material. It is readily possible, however, to use a feedback robot system in which the feedback is provided by sensors.
By the use of the vacuum it is possible to transport fluid, in two steps well defined in time, from one filter into, for example, a second filter arranged below the first in order to be sucked through the second filter into the lowermost region and disposed of.
The invention, especially the vacuum chamber with its at least two vacuum regions which are independent of one another, is not, however, restricted to the transport of fluid in the filtering phase of a plasmid preparation. Other possible uses are the separation of mixtures, the initiation of reactions, the establishment of adsorption processes by the automation (expressed in general terms) of the directed transport of a substance by the provision of at least two vacuum regions that can be established independently of one another. xe2x80x9cSubstancexe2x80x9d is here to be understood as a single substance or a mixture of substances in the form of a fluid, gas or fumes. Furthermore, not only filter supports can be used as the permeable means but the use of, for example, an array of miniaturised chromatography columns is likewise possible, so that time-resolved transport of a substance can also be achieved.